A simple protocol to characterize bacterial cell‐envelope lipoproteins in a native‐like environment

Abstract

Physiological conditions in living cells are strictly regulated to allow, optimize, and coordinate biological processes. The bacterial cell envelope is the compartment where the communication with the external environment takes place. This involves membrane proteins, key players in many biological processes that ensure bacterial survival. The biochemical characterization of membrane proteins, either integral, lipidated or peripheral is challenging due to their mixed protein‐lipid nature, making it difficult to purify and obtain considerable amounts of samples. In contrast to integral membrane proteins, lipidated proteins are usually purified as truncated soluble versions, neglecting the impact of the membrane environment. Here we report a simple and robust protocol to characterize bacterial lipidated proteins in spheroplasts from Escherichia coli using a β‐lactamase as a model. The Metallo‐β‐lactamase NDM‐1 is an enzyme anchored to the inner leaflet of the outer membrane of Gram‐negative bacteria. Kinetic parameters and stability of the lipidated NDM‐1 and the soluble unbound version (NDM‐1 C26A) were measured in spheroplasts and periplasm, respectively. These studies revealed that membrane anchoring increases the KM of the enzyme, consequently decreasing the catalytic efficiency, while not affecting its kinetic stability. This approach can be used to characterize lipidated proteins avoiding the purification step while mimicking its native environment. This approach also helps in filling the gap between in vitro and in vivo studies.

Metallo-β-lactamases in the Age of Multidrug Resistance: From Structure and Mechanism to Evolution, Dissemination, and Inhibitor Design

“…Bacterial spheroplasts are devoid of soluble, periplasmic lactamases but retain the membrane-bound NDM-1 . These preparations can be exploited to retrieve reliable biochemical and biophysical parameters, especially when the protein–membrane interactions are expected to affect the enzyme performance . An alternative, more controlled approach includes the insertion of recombinant lipidated MBLs in artificial liposomes.…”

“…421 These preparations can be exploited to retrieve reliable biochemical and biophysical parameters, especially when the protein−membrane interactions are expected to affect the enzyme performance. 1245 An alternative, more controlled approach includes the insertion of recombinant lipidated MBLs in artificial liposomes. This strategy has allowed the identification of specific interactions between the globular domain of NDM-1 and the bacterial outer membrane.…”

Spheroplasts preparation boosts the catalytic potential of a terpene cyclase

“…1). 29 Under the microscope, the resulting spheroplasts showed the typical, more circular shape, confirming the loss of the outer membrane. 30 The presence of active AacSHC within the spheroplasts was confirmed by SDS-PAGE analysis and activity test (Fig.…”

“…Preparation of spheroplasts. Based on previous protocols, 29,43 100 mg harvested or lyophilized cells were resuspended in 1 mL of 20 mM citric acid at pH 6.0 with 10% sucrose and 150 mM NaCl. After centrifugation (15000 g, 3 min), the cells were resuspended in 20 mM citric acid at pH 6.0 with 10% sucrose, 1 mM EDTA and 1 mg/mL of lysozyme.…”